Mahendra K. Jain

Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z

Actinobacillus sp. 130Z fermented glucose to the major products succinate, acetate, and formate. Ethanol was formed as a minor fermentation product. Under CO2-limiting conditions, less succinate and more ethanol were formed. The fermentation product ratio remained constant at pH values from 6.0 to 7.4. More succinate was produced when hydrogen was present in the gas phase. Actinobacillus sp. 130Z grew at the expense of fumarate and l-malate reduction, with hydrogen as an electron donor. Other substrates such as more-reduced carbohydrates (e.g., d-sorbitol) resulted in higher succinate and/or ethanol production. Actinobacillus sp. 130Z contained the key enzymes involved in the Embden-Meyerhof-Parnas and the pentose-phosphate pathways and contained high levels of phosphoenolpyruvate (PEP) carboxykinase, malate dehydrogenase, fumarase, fumarate reductase, pyruvate kinase, pyruvate formate-lyase, phosphotransacetylase, acetate kinase, malic enzyme, and oxaloacetate decarboxylase. The levels of PEP carboxykinase, malate dehydrogenase, and fumarase were significantly higher in Actinobacillus sp. 130Z than in Escherichia coli K-12 and accounted for the differences in succinate production. Key enzymes in end product formation in Actinobacillus sp. 130Z were regulated by the energy substrates.

Production of butanol and ethanol from synthesis gas via fermentation

The CO strain of Butyribacterium methylotrophicum is able to convert carbon monoxide directly to butanol, ethanol, butyric acid, and acetic acid. The relative proportions of these products can be manipulated by adjusting the fermentation pH. B. methylotrophicum is also tolerant of sulphur-containing gases that can poison inorganic catalysts. Based on these novel metabolic capabilities, one- and two-stage fermentation processes are being developed for production of butanol and ethanol from coal-derived synthesis gas. In the single-stage process, fermentation conditions favouring alcohol formation by B. methylotrophicum are used. In the two-stage process, conditions favouring butyric and acetic acid production are used, and the acids are then reduced to the corresponding alcohols by C. acetobutylicum in a second fermentation.

Evidence for Production of n-Butanol from Carbon Monoxide by Butyribacterium methylotrophicum

Abstract Biological conversion of synthesis gas, primarily a mixture of carbon monoxide (CO) and hydrogen gases, is a potential alternative to chemical processing for production of fuels and chemicals. In addition to acetate and butyrate as metabolic end products, Butyribacterium methylotrophicum has now exhibited n-butanol production directly from CO gas. A butanol concentration as high as 2.7 g/l has been achieved using the CO strain of this organism. These findings suggest the existence of a unique metabolic pathway for butanol production from CO in this strain.

Microbial composition and characterization of prevalent methanogens and acetogens isolated from syntrophic methanogenic granules

The microbial species composition of methanogenic granules developed on an acetate-propionate-butyrate mixture was characterized. The granules contained high numbers of adhesive methanogens (1012/g dry weight) and butyrate-, isobutyrate-, and propionate-degrading syntrophic acetogens (1011/g dry weight), but low numbers of hydrolytic-fermentative bacteria (109/g dry weight). Prevalent methanogens in the granules included: Methanobacterium formicicum strain T1N and RF, Methanosarcina mazei strain T18, Methanospirillum hungatei strain BD, and a non-filamentous, bamboo-shaped rod species, Methanothrix/Methanosaeta-like strain M7. Prevalent syntrophic acetogens included: a butyrate-degrading Syntrophospora bryantii-like strain BH, a butyrate-isobutyrate degrading non-spore-forming rod, strain IB, a propionate-degrading sporeforming oval-shaped species, strain PT, and a propionate-degrading none-spore-forming sulfate-reducing rod species, strain PW, which was able to grow syntrophically with an H2-utilizing methanogen. Sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, acetate and butyrate but they were involved in propionate degradation.

Continuous production of mixed alcohols and acids from carbon monoxide

Continuous, steady-state fermentations using carbon monoxide gas as the sole carbon and energy source have been achieved with the CO strain ofButyribacterium methylotrophicum. Fermentation pH was found to regulate carbon monoxide metabolism over the pH range of 6.8 to 5.0. Cell growth diminished at low pH, with washout occurring at pH 5.0. As observed previously in batch culture, lower pH values favored production of butyrate over acetate. The mechanism responsible for this trend is currently being investigated by quantification of key intracellular enzyme activities.At low pH values, direct, steady-state fermentation of carbon monoxide to alcohols has been verified. Of major significance is the production of butanol from carbon monoxide in pure culture. This newly identified pathway provides a potential mechanism for direct bioconversion of synthesis gas to butanol.

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H2−CO2. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H2 released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H2 plus HCOinf3sup-or produced H2 from formate to a steady-state level at which point the Gibbs free energy (ΔG′) available for formate synthesis or H2 production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H2 pressure. The relative rates of conversion of formate and H2 were apparently controlled by the ΔG′ available for formate synthesis, hydrogen production, methane production from formate and methane production from H2. Results from 14C-tracer tests indicated that a rapid isotopic exchange between HCOO- and HCOinf3sup-occurred during the growth of M. formicicum on H2−CO2. Data from metabolism of 14C-labelled formate to methane suggested that formate was initially split to H2 and HCOinf3sup-and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H2−CO2 was inhibited although production of methane was not Formate synthesis from H2 was also inhibited.

Metabolic properties and kinetics of methanogenic granules

Two types of mesophilic methanogenic granules (R- and F-granules) were developed on different synthetic feeds containing acetate, propionate and butyrate as major carbon sources and their metabolic properties were characterized. The metabolic activities of granules on acetate, formate and H2-CO2 were related to the feed composition used for their development. These granules performed a reversible reaction between H2 production from formate and formate synthesis from H2 plus bicarbonate. Both types of granules exhibited high activity on normal and branched volatile fatty acids with three to five carbons and low activity on ethanol and glucose. The granules performed a reversible isomerization between isobutyrate and butyrate during butyrate or isobutyrate degradation. Valerate and 2-methylbutyrate were produced and consumed during propionate-butyrate degradation. The respective apparent Km (mm) for various substrates in disrupted R- and F-granules was: acetate, 0.43 and 0.41; propionate, 0.056 and 0.038; butyrate, 0.15 and 0.19; isobutyrate, 0.12 and 0.19; valerate, 0.15 and 0.098. Both granules had an optimum temperature range from 40 to 50° C for H2-CO2 and formate utilization and 40° C for acetate, propionate and butyrate utilization and a similar optimum pH.

Biochemical basis for carbon monoxide tolerance and butanol production by Butyribacterium methylotrophicum

Abstract The biochemical mechanisms for growth tolerance to a 100% CO headspace in cultures, and butanol plus ethanol production from CO by Butyribacterium methylotrophicum were assessed in the wild-type and CO-adapted strains. The CO-adapted strain grew on glucose or CO under a 100% CO headspace, whereas, the growth of the wild-type strain was severely inhibited by 100% CO. The CO-adapted strain, unlike the wild-type, also produced butyrate, from either pyruvate or CO. The CO-adapted strain was a metabolic mutant having higher levels of ferredoxin–NAD oxidoreductase activity, which was not inhibited by NADH. Consequently, only the CO-adapted strain can grow on CO because CO oxidation generates reduced ferredoxin which, via the mutated ferredoxin–NAD reductase activity, forms reduced NADH required for catabolism. When the CO-adapted strain was grown at pH 6.0 it produced butanol (0.33 g/l) and ethanol (0.5 g/l) from CO and the cells contained the following NAD-linked enzyme activities (μmol min−1 mg protein−1): butyraldehyde dehydrogenase (227), butanol dehydrogenase (686), acetaldehyde dehydrogenase (82) and ethanol dehydrogenase (129).

Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH

Abstract Exogenous H2/CO2 and glucose were consumed simultaneously by Butyribacterium methylotrophicum when grown under glucose-limited conditions. CO2 reduction to acetate was coupled to H2 consumption. The addition of either H2 or CO2 to glucose batch fermentation resulted in an increase in cell density, hydrogenase (H2-consuming and -producing) activities and fatty acid production by B. methylotrophicum as compared to when N2 was the feed gas. Hydrogenase activities appeared to be tightly regulated and were produced at higher rates during the exponential phase when CO2 was the feed gas as compared to H2 or N2. The increase in H2-consuming activity and decrease in H2-producing activity was correlated with an increase in butyrate synthesis. H2-consuming and ferredoxin (Fd)–NAD reductase activities increased while H2-producing and NADH–Fd reductase activities decreased in cells grown at pH 5.5 compared to those at pH 7.0. The molar ratio of butyrate/acetate was shifted from 0.35 at pH 7.0 to 1.22 at pH 5.5. The addition of exogenous H2 did not decrease the butyrate/acetate ratio at pH 7.0 nor at pH 5.5. The results indicated that growth pH values regulated both hydrogenase and Fd–NAD oxidoreductase activities such that, at acid pH, more intermediary electron flow was directed towards butyrate synthesis than H2 production.

Dechlorination of polychlorinated biphenyl congeners by an anaerobic microbial consortium

Abstract  An anaerobic methanogenic microbial consortium, developed in a granular form, exhibited extensive dechlorination of defined polychlorinated biphenyl (PCB) congeners. A 2,3,4,5,6-pentachlorobiphenyl was dechlorinated to biphenyl via 2,3,4,6-tetrachlorobiphenyl, 2,4,6-trichlorobiphenyl, 2,4-dichlorobi-phenyl and 2-chlorobiphenyl (CB). Removal of chlorine atoms from all three positions of the biphenyl ring, i.e., ortho, meta and para, was observed during this reductive dechlorination process. Biphenyl was identified as one of the end-products of the reductive dechlorination by GC-MS. After 20 weeks, the concentrations of the dechlorination products 2,4,6-CB, 2,4-CB, 2-CB and biphenyl were 8.1, 41.2, 3.0 and 47.8 μM respectively, from an initial 105 μM 2,3,4,5,6-CB. The extent and pattern of the dechlorination were further confirmed by the dechlorination of lightly chlorinated congeners including 2-CB, 3-CB, 4-CB, 2,4-CB and 2,6-CB individually. This study indicates that the dechlorination of 2,3,4,5,6-CB to biphenyl is due to ortho, meta and para dechlorination by this anaerobic microbial consortium.